RU2739302C2 - Antisense compositions and methods for production and use thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions concerns an oral dosage form comprising an antisense oligonucleotide against SMAD7 or a pharmaceutically acceptable salt thereof and an enteric coating, containing an ethyl acrylate-methacrylic acid copolymer. Group of inventions also relates to a method of treating chronic inflammatory bowel disease involving administering said dosage form to a patient; method of delivering the antisense oligonucleotide against SMAD7 to the terminal ileal and ascending colon including administering said dosage form to the patient.

EFFECT: group of inventions provides delivery of an antisense compound to a terminal ileal and colonic ascending.

28 cl, 7 tbl, 9 ex, 7 dwg

Description

This application claims priority on the basis of EP 08425727.8, filed November 12, 2008 and U.S.S.N 61/152297, filed February 1, 2009, both of which are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Ulcerative colitis and Crohn's disease are the main forms of chronic inflammatory bowel disease (IBD) in humans. Intestinal disease of the digestive tract is an inappropriate immune response that occurs in genetically susceptible individuals as a result of a complex interaction between environmental factors, microbial factors and the intestinal immune system. An excessive immune response to mucosal antigens, inadequately regulated by normal antagonistic mechanisms, has been shown to lead to chronic intestinal inflammation.

Crohn's disease is a chronic, recurrent disease of the gastrointestinal tract characterized by segmental transmural inflammation and granulomatous changes. Typical representations include the intermittent involvement of different parts of the gastrointestinal tract and the development of complications including strictures, abscesses, or fistulas. Because its cause is unknown, drug treatments for Crohn's disease are mostly empirical and are designed to reduce inflammation. Drug therapy includes corticosteroids, antibiotics, immunosuppressants, and anti-TNFα agents. Because of the therapeutic setbacks and serious side effects of these therapies, alternatives are needed.

An important role in the pathogenesis of IBD is played by TGF-β1, a multifunctional cytokine capable of regulating the growth, differentiation and function of immune and non-immune cells. The reduced ability to provide an effective TGF-β1 antagonist response to inflammatory stimuli is believed to be significant for the pathogenesis of this IBD disease. TGF-β1 acts as a strong negative regulator of mucosal inflammation, and this suppression of its activity leads to the development of colitis, which has shown an immunomorphological similarity with Crohn's disease or ulcerative colitis.

In the inflamed intestine of IBD patients, there is a marked overexpression of Smad7 (a protein that serves as substrates for TGF-β1 receptors), and a decrease in the phosphorylation of Smad 3, a crucial step in TGF-β1-mediated signal transduction. Thus, in IBD, high levels of Smad7 can lead to defective signal transduction leading to overexpression of genes for proinflammatory molecules, and TGF-β1 does not show its anti-inflammatory role.

Antisense oligodeoxynucleotide drugs are short chains of DNA nucleotides that inhibit protein translation by specifically binding to a small segment of messenger RNA (mRNA) responsible for stimulating the production of disease-causing proteins. The sequence of the antisense drug is designed to be complementary to its target mRNA, so that upon hybridization, the resulting double-stranded segment is recognized by the cell as abnormal and destroyed, thereby preventing translation of information into the protein product.

Antisense therapeutic agents, however, are usually administered parenterally, which can lead to adverse reactions due to systemic effects. With this introduction, it is also impossible to localize the drug at the site of the required treatment. Therefore, there is a need for the use of antisense treatments like topical treatments for IBD and related diseases using tablet formulations.

SUMMARY OF THE INVENTION

This disclosure relates, at least in part, to pharmaceutical compositions for oral administration of antisense oligonucleotides, such as anti-SMAD7 antisense oligonucleotides.

In one embodiment, there is provided a pharmaceutical tablet formulation for oral administration of an antisense oligonucleotide, which comprises an intragranular phase, wherein the intragranular phase comprises an antisense oligonucleotide such as represented by SEQ ID NO 1, or a pharmaceutically acceptable salt thereof (such as sodium salt) and a pharmaceutically acceptable excipient, and which may also include an extragranular phase, which may include a pharmaceutically acceptable excipient such as a disintegrant. Contemplated oligonucleotides include those represented by SEQ ID NO 1, wherein at least one, or in some embodiments, all internucleotide linkages are O, O-linked phosphorothioates.

This description discloses a tablet that includes the disclosed antisense oligonucleotide and contains an enteric coating. Such a tablet may, for example, include a filler, disintegrant and / or lubricant. For example, provided herein is an oral dosage form, such as a tablet, that contains about 35 mg to about 500 mg of an antisense oligonucleotide, eg, 40 mg of an oligonucleotide represented by SEQ ID NO 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, there is provided an oral tablet comprising: from about 0.5 to about 10% by weight of the antisense oligonucleotide represented by SEQ ID NO 1 or a pharmaceutically acceptable salt thereof; from about 30% to about 50% by weight of mannitol; and from about 10% to about 30% by weight of microcrystalline cellulose.

For example, this disclosure provides a pharmaceutically acceptable tablet for oral use comprising an intragranular phase and an extragranular phase, whereby, for example, the intragranular phase contains from about 5% to about 10% by weight (e.g., from about 8% by weight) of the antisense oligonucleotide represented by SEQ ID NO 1 or a pharmaceutically acceptable salt thereof, about 40% by weight of mannitol, about 8% by weight of microcrystalline cellulose, about 5% by weight of hydropropyl methylcellulose and about 2% by weight of sodium starch glycolate and, for example, the extragranular phase contains about 17% by weight microcrystalline cellulose, about 2% by weight sodium starch glycolate and about 0.4% by weight magnesium stearate, where the tablet may further include an enteric coating.

Also provided herein are methods of treating Crohn's disease, ulcerative colitis and chronic inflammatory bowel disease, comprising administering to a patient in need thereof a tablet, oral dose, or pharmaceutical formulation described herein. For example, when a pharmaceutical formulation, tablet, or oral dosage form is orally administered to a patient, the pharmaceutical formulation, tablet, or oral dosage form may be primarily delivered to the terminal ileum and / or to the ascending colon of the patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the molecular structure of an antisense compound described herein as AS1.

FIG. 2 is a schematic representation of the manufacturing process of AS1.

FIG. 3 is a bar graph representing particle size distribution for a 3.5 mg dose of AS1.

FIG. 4 is a bar graph representing the particle size distribution for a dose of 35 mg AS1.

FIG. 5 is a bar graph representing the particle size distribution for a 250 mg dose of AS1.

FIG. 6 is a line graph representing dissolution patterns (as percentage of tablet dissolved) for three tablets at different dosage levels of AS1 in three different media (pH 1.0, pH 6.6, and pH 7.2).

FIG. 7 represents the dissolution behavior of the batch described herein.

DETAILED DESCRIPTION OF THE INVENTION

This description generally refers to pharmaceutical compositions that include an antisense oligonucleotide such as shown in FIG. 1. Contemplated compositions include oligonucleotides that act against Smad7 and can be administered orally. The disclosed compositions, when administered orally, can deliver an effective amount of an antisense oligonucleotide to the intestinal system of a patient, for example, deliver an effective amount of an antisense oligonucleotide to the terminal ileum and / or ascending colon of a patient.

Contemplated antisense oligonucleotides include those containing SEQ ID NO 1 GTC * GCC CCT TCT CCC C * GC AGC, where C * is 5-methyl-2'-deoxycytidine. In some embodiments, at least one of the internucleotide linkers of the antisense oligonucleotide in question is O, O-linked phosphorothioates, for example, each of the 20 internucleotide linkers of SEQ ID NO 1 may be O, O-linked phosphorothioate. In some embodiments, the contemplated compositions described herein may include a pharmaceutically acceptable salt, eg, the sodium salt of the antisense oligonucleotide SEQ ID NO 1, which optionally may include from 1 to 20 O, O-mediated phosphorothioate internucleotide linkers. Contemplated salts of oligonucleotides include salts that are completely neutralized, for example, each phosphorothioate linker is linked to an ion such as Na ⁺ . Oligonucleotides can include naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as non-naturally occurring moieties. An example of an antisense oligonucleotide, referred to herein as AS1, is shown in FIG. one.

In some embodiments contemplated herein, compositions are provided that are suitable for oral delivery of an antisense oligonucleotide, for example, tablets that include an enteric coating, such as a stomach-stable coating, such that the compositions can deliver the antisense compound, for example, to the terminal iliac region. the intestine and the ascending colon of the patient. For example, the route of administration can result in a local action, essentially local application of the antisense compound to the affected part of the intestine of a patient. Such use, in some embodiments, may substantially avoid undesirable systemic absorption of the antisense compound.

For example, a tablet for oral administration is provided that contains granules (eg, is at least partially formed from granules) that include an antisense compound, eg AS1, and pharmaceutically acceptable excipients. Such a tablet can be enteric coated. The contemplated tablets may include pharmaceutically acceptable excipients such as fillers, binders, disintegrants and / or lubricants, as well as release-enhancing colorants, coating agents, sweeteners, flavors such as wintergreen, orange, xylitol, sorbitol, fructose and maltodextrin; and flavors, preservatives and / or antioxidants.

In some embodiments, contemplated pharmaceutical formulations comprise an intragranular phase that includes an antisense compound of interest, eg, that depicted in SEQ ID NO 1, or a pharmaceutical salt thereof, eg AS1, and a pharmaceutically acceptable excipient. For example, AS1 and filler can be mixed with each other, with optionally other excipients, and formed into granules. In some embodiments, the intragranular phase can be formed using wet granulation, for example, a liquid (eg, water) is added to the miscible antisense compound and excipient, and then the combination is dried, milled and / or sieved to form granules. One skilled in the art should understand that other processes can be applied to obtain the intragranular phase.

In some embodiments, the contemplated formulations include an extragranular phase, which may include one or more pharmaceutically acceptable excipients, and which may be mixed with the intragranular phase to form the disclosed composition.

The described composition may include an intragranular phase that includes a filler. Examples of fillers include, but are not limited to, cellulose, gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, pectin, polyacrylates, dectrose, cellulose acetate, hydroxypropyl methylcellulose, partially pregelatinized starch, calcium carbonate, and others ...

In some embodiments, the composition described may include an intragranular phase and / or an extragranular phase that includes a binder that can normally function to hold the ingredients of the pharmaceutical composition together. Examples of binders included in the invention include, but are not limited to, the following: various types of starch, sugar, cellulose or modified cellulose such as hydroxypropyl cellulose, lactose, pregelatinized corn starch, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium hydroxypropyl cellulose, low-cellulose cellulose methylcellulose, ethylcellulose, sugar-alcohols, and other combinations included therein.

Contemplated formulations, for example, those that include an intragranular phase and / or an extragranular phase, may include a disintegrant such as, but not limited to, starch, cellulose, structured polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, carboxymethylcellulose, alginocellulose, granular hydroxypropyl cellulose, acacia gum, and other combinations thereof. For example, the intragranular phase and / or extragranular phase may include a disintegrant.

In some embodiments, the composition under consideration includes an intragranular phase containing the described antisense compound and excipients selected from: mannitol, microcrystalline cellulose, hydroxypropyl methylcellulose, and sodium starch glycolate or combinations thereof, and an extragranular phase containing one or more of: microcrystalline cellulose and starch glycol, sodium their mixtures.

In some embodiments, the contemplated formulation may include a lubricant, for example, the extragranular phase may contain a lubricant. Lubricants include, but are not limited to, talc, silica, fats, stearin, magnesium stearate, calcium phosphate, silicon dioxide, calcium silicate, calcium phosphate, colloidal silicon dioxide, metal stearates, hydrogenated vegetable oil, grain starch, sodium benzoate, polyethylene glycols , sodium acetate, calcium stearate, sodium lauryl sulfate, sodium chloride, magnesium lauryl sulfate and stearic acid.

In some embodiments, the pharmaceutical composition includes an enteric coating. Typically, enteric coatings provide a barrier to oral drug formulations that regulates the localization at which the drug is absorbed in the digestive tract. Enteric coatings can include a polymer that degrades to varying degrees according to pH. Enteric coatings may include, for example, cellulose acetate phthalate, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, methyl methacrylate-methacrylic acid copolymers, ethyl acrylate-methacrylic acid copolymers, cellulose acetate polymers, methacrylic acid copolymer, cellulose acetate copolymer, copolymer of methacrylic acid copolymer, and cellulose acetate copolymer.

Examples of enteric coatings include Opadry ^® AMB ^{brand, Acryl-EZE ®, Eudragit ®} . In some embodiments, the enteric coating may be from about 5% to about 10%, from about 5% to about 20%, from 8 to about 15%, from about 8% to about 18%, from about 10% to about 12%, from about 12% to about 16% of the considered tablet by weight. For example, enteric coatings can include an ethyl acrylate-methacrylic acid copolymer.

For example, a tablet is provided that includes or contains substantially from about 0.5% to about 70%, for example, from about 0.5% to about 10%, or from about 1% to about 20%, by weight of an antisense oligonucleotide or a pharmaceutically acceptable salt (eg AS1). Such a tablet may include, for example, from about 0.5% to about 60% by weight of mannitol, for example, from about 30% to about 50% by weight of mannitol, for example, about 40% by weight of mannitol; and / or from about 20% to about 40% by weight of microcrystalline cellulose or from about 10% to about 30% by weight of microcrystalline cellulose. For example, the disclosed tablet may contain an intragranular phase that comprises from about 30% to about 60%, for example, from about 45% to about 65% by weight, or alternatively from about 5% to about 10% by weight AS1, from about 30% to about 50%, or alternatively from about 5% to about 15%, by weight of mannitol, from about 5% to about 15% microcrystalline cellulose, from about 0% to about 4%, or from about 1% to about 7% hydroxypropyl methylcellulose; and about 0% to about 4%, for example, about 2% to about 4% sodium starch glycolate, by weight.

Examples of formulations include dosage forms that include or contain from about 35 to about 500 AS1, for example, tablets are contemplated that include about 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg , 150 mg, 200 mg or 250 mg AS1.

In one embodiment, a pharmaceutically acceptable tablet for oral use is provided that includes an intragranular phase that may contain about 50% by weight AS1 (or a salt thereof), about 11.5% by weight mannitol, about 10% by weight microcrystalline cellulose, about 3% by weight of hydropropyl methylcellulose and about 2.5% by weight of sodium starch glycolate; and an extragranular phase which may contain about 20% by weight of microcrystalline cellulose, about 2.5% by weight of sodium starch glycolate and about 0.5% by weight of magnesium stearate. The tablet may also include an enteric coating.

In another embodiment, there is provided a pharmaceutically acceptable tablet for oral use, which comprises or consists essentially of: an intragranular phase which may contain or essentially consist of from about 5% to about 10%, for example, from about 8% by weight AS1 (for example, in which internucleotide binders are each O, O-linked phosphorothioates and / or a salt thereof, for example, sodium salt), about 40% by weight of mannitol, about 8% by weight of microcrystalline cellulose, about 5% by by weight of hydropropyl methylcellulose and about 2% by weight of sodium starch glycolate; and an extragranular phase which may contain about 17% by weight of microcrystalline cellulose, about 2% by weight of sodium starch glycolate and about 0.4% by weight of magnesium stearate.

The lozenges described herein may also comprise an enteric coating, for example, describes a tablet may include about 13%, about 15%, 16%, 17% by weight of the enteric coating, for example, AcyrlEZE ^®.

The rate at which the coating dissolves and the active ingredient is released is an indicator of its dissolution. In one embodiment, a subject tablet may have dissolution rates, for example, when tested in a USP / EP Type 2 apparatus (paddle mixer) at 100 rpm and 37 ° C in phosphate buffer at pH 7.2 from 50% to about 100% release of the oligonucleotide after about 120 minutes to about 240 minutes, for example after 180 minutes. In another embodiment, the tablet in question may have dissolution characteristics, for example when tested in a USP / EP Type 2 apparatus (paddle mixer) at 100 rpm and 37 ° C in dilute HCl pH 1.0, when the oligonucleotide is substantially not released after 120 minutes. In yet another embodiment, the tablet in question may have dissolution rates, for example, when tested in a USP / EP Type 2 paddle mixer at 100 rpm and 37 ° C in phosphate buffer pH 6.6 from about 10% to about 30% or not more than about 50% release of the oligonucleotide after about 30 minutes.

The disclosed formulations, eg, tablets, in some embodiments, when taken orally by a patient, may result in minimal plasma oligonucleotide concentrations in the patient. In another embodiment, the disclosed formulations, when administered orally to a patient, are locally delivered to the terminal ileum and / or ascending colon of the patient, for example, at a diseased or diseased site of the patient's intestine.

Also provided herein are methods for treating Crohn's disease, ulcerative colitis and / or chronic inflammatory bowel disease in a patient in need of this use of the disclosed composition.

EXAMPLES

The examples that follow are not intended in any way to limit the scope of this invention, but are presented to illustrate the methods of this invention. Many other embodiments of this invention will be apparent to a person skilled in the art.

Example 1. Tablets

Wet granules were prepared by dispensing each intragranular component into an appropriate container. All intergranular substances were sieved through a 710 μm sieve and mixed in a food processor bowl for about 5 minutes. The aqueous granulation liquid was added slowly using a syringe. The wet mass was passed through a 2.00 mm hand sieve and dried in a thermostat at 40 ° C for up to 90 minutes. After drying, the granules were sieved through a 1.00 mm sieve. A mortar and pestle were used to reduce the size of the large granules. The granules were blended with intragranular excipients, excluding magnesium stearate, using a Turbula blender for 10 minutes at 42 rpm. Magnesium stearate was added to the mixture and further stirred for 2 minutes at 42 rpm. The compositions were pressed on a Manesty F3 single punch press machine. The general flow chart for the AS1 tablets can be seen in FIG. 2.

A 250 mg mannitol-based formulation made in a 50 g batch had a dry mix moisture content of 5.21% and a dried granule moisture content of 6.42%. The mannitol-based formulation at the 250 mg dosage level had 5.0 g of added water, the granulation time was 4 minutes and the drying time was 60 minutes.

The 35 mg mannitol-based formulation made in a 100 g batch had a moisture content of 2.35% for the dried granules, 28 g of water was added, the granulation time was 6 minutes and the drying time was 65 minutes.

IPC compaction results in 250 mg mannitol-based formulations that are: average weight 453.0 mg, hardness 18.0 kp, thickness 3.82 mm, friability 0.23% and disintegration 17 minutes.

Table 1A includes compositions for three dose levels: 3.5 mg, 35 mg and 250 mg. Tablet weight was 500 mg for all formulations.

Table 1B presents the composition of batches of kernels at these three dosage levels:

Example 2. Characteristics of the powder

The formulations were evaluated for particle size distribution, density, Kara index and angle of repose. Particle size distribution analysis showed that the particles were larger (335 μm) for the 3.5 mg dosage level formulation, while the 35 mg and 250 mg dosage level formulations exhibited a typical particle size distribution. FIG. 3 shows the particle size distribution for a 3.5 mg dosage at the formulation shown in Table 1. FIG. 4 shows the particle size distribution for the 35 mg dosage at the formulation shown in Table 1. Finally, FIG. 5 shows the particle size distribution for the 250 mg dosage with the formulation shown in Table 1. Table 2 shows the powder characterization results for the three dosage levels.

Example 3 - Enteric Coating

From Acryl-EZE ^® coatings prepared 2 0% coating solution. The required amount of water and Acryl-EZE ^® metered into appropriate containers. With stirring, Acryl-EZE ^® slowly added into the vortex. The dispersion was stirred for 45 minutes and passed through a 500 μm sieve. Spraying was continued until a weight gain of 10% or 16% was obtained.

Example 4 - 40 mg tablet

The batch composition of 40 mg AS1 tablets is shown below.

Example 5 - Composition of 200 mg tablets

AS1 tablets with a dosage level of 200 mg were produced essentially according to the procedure of Example 1, as shown in Table 4. The tablet weight for all formulations was 500 mg.

Example 6. Analysis of dissolution by HPLC

The analytical test procedure describes the dissolution analysis of enteric coated tablets AS1 by HPLC. Dissolution is carried out according to the method of the European Pharmacopoeia for solid dosage forms with delayed release using method A. The apparatus used was apparatus 2 (paddle mixer) according to Eur. Pharmacopoeia / United States Pharmacopoeia.

Dissolution conditions for HPLC are as follows: medium parameters are: pH 1.0 HCl (120 min), pH 6.6 (30 min), pH 7.2 (60 min); temperature 37 ° C; speed 100 rpm; sample for recirculation 7.5 ml, sample size 0.8 ml; processing time of the sample 120 min in HCl at pH 1.0; 15, 30 min at pH 6.6; 15, 30, 45, 60 min at pH 7.2; and a 45 micron Disteck inline filter.

The medium in each vessel was adjusted during dissolution in each state as follows: an initial volume of 750 ml HCl pH 1.0 was added at 120 min; 200 ml 0.2 M Na ₃ PO ₄ and 30 ml 1.0 M Na ₂ HPO ₄ pH 6.7 followed by adjusting the pH to 6.60 ± 0.05 with 2.0 M NaOH; at 150 min, followed by adjusting the pH to 7.20 ± 0.05 with 2.0 M NaOH.

The chromatographic conditions are as follows: Dionex analytical HPLC column, DNAPPac-100, 4 x 250 mm; flow rate 2.0 ml / min; column temperature 80 ° C; UV detection at 260 nm; injection volume 100 μl; rinsing the needle with water; mobile phase A) 10% (v / v) acetonitrile in 100 mM Tris (pH 8.0) and B) 10% (v / v) acetonitrile in 100 mM Tris and 2M LiCl (pH 8.0); and an HPLC cycle time of 15 min; and an AS1 elution period of about 6 minutes. The gradient is shown in Table 6.

Preparation of 35 mg tablet working standard solutions: 12.5 mg of AS1 reference standard was placed in a 50 ml flask and dissolved in water to prepare a 50 ml solution. 7 ml of the solution was diluted with water to make 50 ml of solution to give a final AS1 concentration of 0.035 mg / ml.

Preparation of standard working tablet solutions 250 mg: 12.5 mg of AS1 reference standard was placed in a 50 ml flask and dissolved in 50 ml water to give a final AS1 concentration of 0.25 mg / ml.

Table 5 shows dissolution results for 35 mg, 250 mg and 200 mg dosage batches and 12% weight gain when coated with Acryl-EZE®. FIG. 6 is a graph of the dissolution characteristics of tablets of AS1 formulations with three dosage levels in three different media.

Tablets coated with 16% Acryl-EZE ^® manufactured using the tablet as described above for Part 1, and the final coating is carried out until when the weight gain of the tablets in a coating pan of 16% of the initial value. FIG. 7 is a dissolution graph.

Example 7. Dosage of AS1 in vivo by oral administration

The objectives of this study were to identify the potential effect of AS1 on the cardiovascular, respiratory and central nervous systems in conscious free-range cynomolgus monkeys when administered in a single dose by oral administration or intravenous administration. Intravenous administration was included in this study to investigate the potential effects associated with systemic exposure. The intravenous dosage formulation was used as a non-packaged powder, but for oral administration in the form of enteric-coated tablets placed in gelatin capsules. Four not for the first time used in the experiment male cynomolgus macaques aged 3.6-3.8 years and weighing 3.4-3.9 kg were identified in one group.

All dosage levels represent the amount of AS1 corrected for purity. Oral control doses were two capsules containing placebo tablets. An approximate dosage level was obtained by administering two capsules, each containing a tablet with 26.1 mg AS1 per tablet (adjusted for purity) and based on body weight, which was in the range of 3.5-3.9 kg on day 4. An approximate dosage level was obtained by administering two capsules, each containing a tablet with 169.9 mg AS1 per tablet (adjusted for purity) and based on body weight, which ranged from 3.5 to 3.9 kg on day 4 ...

Blood samples for toxicokinetic analysis were taken before the study and 1 and 6 hours after oral administration of the drug and 5 minutes, 1 hour and 6 hours after intravenous administration of the drug. The samples were processed to obtain plasma under refrigeration conditions, and the plasma was stored at -70 ° C until analysis. Samples were analyzed using an assay for specific hybridization with AS1.

On day 1, all animals received the orally named control dose followed by doses of AS1 on days 4 and 7 via oral gavage. For oral doses, placebo and AS1 tablets were enclosed in gelatin capsules of appropriate size to facilitate oral administration. On day 12, all animals received an intravenous control dose (phosphate buffered saline) by slow injection through a syringe followed by intravenous doses of AS1 on days 14 and 19.

Data on cardiovascular system and body temperature were recorded by telemetry frequently, at short intervals before administration and within 24 hours after administration of each dose. Respiratory function was assessed by measuring blood gas parameters in arterial blood samples taken before administration and at 1, 6 and 24 hours after each dose, as well as by measuring respiration rate (visually) at the same time points. Neurological function was assessed by performing a comprehensive neurological examination of the animals before the study and within about 2 to 4 hours after the end of the telemetry recording period after the last oral dose of the test drug, and again within about 24 hours after the end of the telemetry recording period after the last intravenous dose. The animals were also monitored for clinical manifestations (mortality / morbidity, side effects chamber observations, food intake and body weight).

Despite the very low LLOQ for analysis (0.5 ng / ml), AS1 was not detected in any plasma samples after oral administration of doses up to about 100 mg / kg. In contrast, the mean maximum plasma concentrations after iv injection were 28.309 and 180.352 ng / ml at doses of 3 and 10 mg / kg, respectively. Clearance of AS1 from plasma after IC dosing exhibited kinetics similar to those reported for structurally related oligonucleotides (ie, half-life of about 0.5 hours).

Example 8 28-Day In Vivo Oral AS1 Study

The aim of this study was to evaluate the potential toxicity and toxicokinetics of AS1 when administered orally once daily to mice for 28 days, followed by a 28-day recovery period. The AS1 formulation was prepared as a formulation containing a stomach protectant coating in the form of small coated granules. AS1 was layered onto inert granules which were then coated with Eudragit® S 100 mimicking oral formulations suitable for use in humans. Three groups were used, which were dosed at various levels (30 mg / kg / day (low); 100 mg / kg / day; 300 mg / kg / day).

There were no gender-related differences in plasma and tissue drug levels. Despite the very low NPAR for this assay, AS1 was only detected in two plasma samples from animals at a very low dose (30 mg / kg / day) taken on day 1, and not in any samples taken on day 28. At higher dose levels of 100 and 300 mg / kg / day, AS1 was detectable in most plasma samples and plasma levels were largely dose dependent. However, even at the highest dose level, plasma levels did not exceed 21 ng / ml in any animal (in samples collected at different times between 0.5 and 24 hours after administration). AS1 concentrations were very high in gastrointestinal tract tissues with average maximum concentrations at the highest dosage level (after the first administration) of about 536, 857, 825, 538, 137 and 127 μg / gram tissue for large intestine, small intestine, cardiac part of the stomach, glandular part of the stomach, esophagus and rectum, respectively.

Intensive elimination from the tissues of the gastrointestinal tract was evident 24 hours after the first dose. There was no apparent accumulation of AS1 with daily administration.

The maximum mean concentrations in the two main organs after systemic administration, kidney and liver, after the first dose of 300 mg / kg were only 4.0 and 2.3 μg / gram, which is 100 times lower than the order of concentrations determined in tissues of the gastrointestinal tract.

After the lowest dose of 30 mg / kg was administered, the highest mean kidney and liver concentrations were only 0.4 and 0.3 μg / gram. There was no data on the accumulation of AS1 in the viscera during the 28 day administration period.

8 последовательных дней.The systemic exposure to AS1 in mice was very low after oral administration of high doses (up to 300 mg / kg / day) of AS1 delivered in a formulation protected from the stomach environment, and there was no accumulation in the gastrointestinal tract tissues in the tissues of internal organs when administered daily for 2

8 consecutive days.

Example 9. Therapeutic action of AS1 in colitis

To evaluate the therapeutic effect of AS1 on the course of intestinal inflammation in a mouse model of TNBS-induced colitis, mice were administered a single dose of AS1 or Smad7 sense oligonucleotide one day after TNBS rectal administration. Single doses of 125 or 250 μg / mouse reduced weight loss and markedly decreased the severity of the histological manifestations of colitis.

AS1 (assuming a single dose of 125 μg once daily after induction of colitis with TNBS) significantly reduced colonic production of the monomeric p40 subunit, a component of IL-12 and IL-23 cytokines, demonstrating suppression of colonic production of these two pro-inflammatory cytokines ...

All publications and patents mentioned herein, including those listed below, are hereby incorporated by reference in their entirety, as if each individual publication or patent were specifically and separately incorporated herein by reference. In the event of a conflict, this application will be monitored, including any definitions provided herein.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the foregoing description is illustrative and not restrictive. Many variations of the present invention will become apparent to those skilled in the art upon consideration of this description. The full scope of this invention is to be determined by reference to the claims in conjunction with the full scope of their equivalents and the description in conjunction with similar options.

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LIST OF SEQUENCES

<110> Giuliani International Limited

BARONI, Sergio

BELLINVIA, Salvatore

VITI, Francesca

<120> Antisense compositions and methods for their preparation and use

<130> P46432WO / JCH

<150> EP08425727.8

<151> 2008-11-13

<150> US61 / 152.297

<151> 2009-02-01

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> Artificial

<220>

<223> oligonucleotide

<220>

<221> modified base

<222> (3) .. (3)

<223> 5-methyl-2'-deoxycytidine

<220>

<221> modified base

<222> (16) .. (16)

<223> 5-methyl-2'-deoxycytidine

<400> 1

gtcgcccctt ctccccgcag c 21

<---

Claims (28)

1. A dosage form for oral administration containing an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof and an enteric coating containing an ethyl acrylate-methacrylic acid copolymer. 2. The dosage form of claim 1, wherein 10% to 30% of the anti-SMAD7 antisense oligonucleotide is released in the pH 6.6 environment from the dosage form within 30 minutes by HPLC dissolution analysis, and the anti-SMAD7 antisense oligonucleotide is substantially not released in an environment with a pH of 1.0 within 120 minutes by HPLC dissolution analysis. 3. The dosage form of claim 1, wherein no more than 50% of the anti-SMAD7 antisense oligonucleotide is released in the environment at pH 6.6 from the dosage form within 30 minutes by HPLC dissolution analysis, and the anti-SMAD7 antisense oligonucleotide is substantially not released in an environment with a pH of 1.0 for 120 minutes when analyzed by HPLC dissolution. 4. A dosage form according to claim 1, wherein the anti-SMAD7 antisense oligonucleotide is not substantially released in a pH 1.0 environment for 120 minutes by HPLC dissolution analysis. 5. The dosage form according to any one of paragraphs. 1-4, in which the anti-SMAD7 antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, contains the nucleotide sequence of SEQ ID NO: 1, wherein all internucleotide linkers of the anti-SMAD7 antisense oligonucleotide are O, O-linked phosphorothioates. 6. Dosage form according to any one of paragraphs. 1-4, wherein the HPLC dissolution analysis is performed at 37 ° C. in a USP / EP Type 2 apparatus with a paddle speed of 100 rpm. 7. A dosage form according to claim 6, wherein the pH 6.6 environment contains phosphate buffer and the pH 1.0 environment contains HCl. 8. The dosage form according to any one of paragraphs. 1-7, with the dosage form being a tablet. 9. A method of treating chronic inflammatory bowel disease, comprising orally administering to a patient in need thereof a dosage form containing an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof and an enteric coating containing an ethyl acrylate-methacrylic acid copolymer. 10. The method of claim 9, wherein 10% to 30% of said amount of anti-SMAD7 antisense oligonucleotide from the dosage form is released in an environment with a pH of 6.6 for 30 minutes by HPLC dissolution analysis, and the anti-SMAD7 antisense oligonucleotide according to essentially not released in a pH 1.0 environment within 120 minutes when analyzed by HPLC dissolution. 11. The method of claim 9, wherein in a pH 6.6 environment no more than 50% of the anti-SMAD7 antisense oligonucleotide is released from the dosage form within 30 minutes by HPLC dissolution analysis, and the anti-SMAD7 antisense oligonucleotide is substantially not released in ambient pH 1.0 for 120 minutes when analyzed by HPLC dissolution. 12. The method of claim 9, wherein the anti-SMAD7 antisense oligonucleotide is not substantially released in a pH 1.0 environment for 120 minutes when analyzed by HPLC dissolution. 13. The method according to any one of claims. 9-12, wherein the anti-SMAD7 antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, contains the nucleotide sequence of SEQ ID NO: 1, wherein all internucleotide linkers of the anti-SMAD7 antisense oligonucleotide are O, O-linked phosphorothioates. 14. The method according to any one of claims. 9-12, wherein the HPLC dissolution analysis is performed at 37 ° C. in a USP / EP Type 2 apparatus with a paddle speed of 100 rpm. 15. The method of claim 14, wherein the pH 6.6 environment contains phosphate buffer and the pH 1.0 environment contains HCl. 16. The method according to any one of claims. 9-15, wherein the dosage form is a tablet. 17. The method according to any one of claims. 9-16, wherein the chronic inflammatory bowel disease is Crohn's disease or ulcerative colitis. 18. The method according to any of paragraphs. 9-17, wherein the dosage form contains 35 mg to 500 mg of an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof. 19. The method according to any one of claims. 9-18, wherein the dosage form is administered orally to a patient to deliver the dosage form substantially to the terminal ileum and / or the ascending colon. 20. A method of delivering an anti-SMAD7 antisense oligonucleotide to at least one of the terminal ileum and / or the ascending colon, the method comprising administering to the patient an oral dosage form containing the anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof and an enteric a coating comprising an ethyl acrylate-methacrylic acid copolymer, wherein an effective amount of an antisense oligonucleotide is delivered to the terminal ileum and the ascending colon of the patient. 21. The method of claim 24, wherein the anti-SMAD7 antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, wherein all internucleotide linkers of the anti-SMAD7 antisense oligonucleotide are O, O-linked phosphorothioates. 22. The method of claim 20, wherein the dosage form contains 35 mg to 500 mg of an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof. 23. A method of releasing an anti-SMAD7 antisense oligonucleotide in the intestinal system of a patient, the method comprising administering to the patient an oral dosage form containing an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof and an enteric coating containing an ethyl acrylate-methacrylic acid copolymer, an effective amount of the anti-SMAD7 antisense oligonucleotide is released in the intestinal system of the patient, and the anti-SMAD7 antisense oligonucleotide is not substantially released in the stomach and the therapeutic effect is carried out in the intestinal system. 24. The method of claim 23, wherein the anti-SMAD7 antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1, wherein all internucleotide linkers of the anti-SMAD7 antisense oligonucleotide are O, O-linked phosphorothioates. 25. The method of claim 23, wherein the dosage form contains 35 mg to 500 mg of an anti-SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof. 26. The method of claim 23, wherein the anti-SMAD7 antisense oligonucleotide is released in the terminal ileum. 27. The method of claim 23, wherein the anti-SMAD7 antisense oligonucleotide is released in the ascending colon. 28. The method of claim 23, wherein administering the dosage form to the patient provides a minimum plasma concentration of the anti-SMAD7 antisense oligonucleotide in the patient.

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